Abstract

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

Interaction between ERdj5 and SERCA2. (A and B) Forty-eight hours after transfection of nonspecific (NS) or SERCA2-specific siRNA into HeLa cells, cell lysates were prepared for immunoprecipitation with an anti-SERCA2 antibody. Immunoprecipitates were subjected to (A) reducing or (B) nonreducing (Left) and reducing (Right) SDS/PAGE for the analysis by immunoblotting with the indicated antibodies. (C) The series of ERdj5 mutants constructed in this work. (D and E) Twenty-four hours after cotransfection of (D) HA-tagged or (E) PA-tagged SERCA2b and FLAG-tagged ERdj5/WT or the indicated ERdj5 mutants into HEK293 cells, cell lysates were prepared for immunoprecipitation with (D) anti-HA or (E) anti-PA antibodies. (D and E) All immunoprecipitates were subjected to reducing SDS/PAGE and analyzed by immunoblotting with the indicated antibodies. The asterisk in D indicates an NS band. CNX, calnexin.

ERdj5/CA4 interacts with SERCA2 through the disulfide bond. (A) Schematic representation of ERdj5/CA4. (B) Twenty-four hours after cotransfection of PA-tagged SERCA2b and FLAG-tagged ERdj5/AA or ERdj5/CA4 mutant into HEK293 cells, cells were treated with DVSF, which is the cross-linker specific for disulfide bond. The lysates were added with 1% SDS to dissociate the noncovalent interaction. After the dilution of SDS to 0.1%, the cell lyates were immunoprecipitated with anti-PA antibodies and analyzed by SDS/PAGE under reducing condition.

Deficiency of ERdj5 suppresses Ca2+ uptake into the ER through SERCA2b. (A and B) Cells were loaded with Mag-Fura-2 to estimate [Ca2+]ER. Semipermeabilized MEFs were treated with EGTA for 20 min to remove Ca2+. After depletion of Ca2+, ATP and Ca2+ were added to stimulate SERCA2 pump functions. Mag-Fura-2 fluorescence was measured as [Ca2+]ER. The quantifications of each Ca2+ uptake are as shown in B. (C and D) After 1 µM thapsigargin treatment, [Ca2+]i was measured using Mag-Fura-2. Peak amplitudes are shown as a bar graph in D.

The expression levels of SERCA2b were unaffected by the overexpression of ERdj5s. Twenty-four hours after transfection of ERdj5 WT, AA mutant, or H63A mutant into ERdj5 (−/−) MEF cells, cell lysates were prepared for immunoblotting with anti-SERCA antibody (Upper) or anti–β-actin antibody (Lower).

ERdj5-knockdown cells are sensitive to ER stress induced by the Ca2+ ionophore A23187. Unfolded protein response induction was examined by detecting the spliced form of XBP1 mRNA. Total RNA was prepared from HEK293T cells 48 h after transfection. Cells treated with 100 nM A23187 for the indicated amounts of time were used as positive controls. Unspliced (U) and spliced (S) forms of hXBP1 mRNA amplified by RT-PCR are shown.

ERdj5 cleaves the disulfide bond of SERCA2b and activates its function. (A) Comparison of ATPase activity between the oxidized and reduced forms of SERCA2b. The ATPase activities of reduced SERCA2b (red line), oxidized SERCA2b (blue line), and rereduced SERCA2b (orange line) were assessed by quantifying released Pi. Results presented in the graphs represent means ± SD of three independent experiments. (B) Activation of the SERCA2b ATPase by ERdj5/WT and the ERdj5/SS mutant. The concentration of Pi released by 50 nM SERCA2b in the presence of 0, 12.5, 25, 50, 125, or 250 nM ERdj5/WT or ERdj5/SS was quantified and plotted as a function of the reaction time. Results presented in the graphs represent means ± SD of three independent experiments. (C) To accurately estimate the redox states of SERCA2b, SERCA2b/KK was constructed, in which two arginines in the C terminus were converted to lysines. After SERCA2b/KK was transfected into HEK293T cells, the cells were precipitated with 10% TCA, and free thiol groups were modified with 150 mM AMS. The C-terminal region containing C875, C887, and the PA tag was excised using Arg-C endopeptidase, which selectively cleaves C-terminal to arginine residues. (D) Forty-eight hours after transfection of nonspecific (NS) or ERdj5-specific siRNA into HeLa cells, cell lysates were prepared for Arg-C treatment as described in C. After immunoprecipitation with anti-PA antibody, a concentrated C-terminal region was detected by Western blotting with an anti-PA antibody.

Interaction between ERdj5 and SERCA2 depending on [Ca2+]. (A) Redox states of ERdj5 under treatment with thapsigargin (Tg) or tunicamycin (Tu). Transfected FLAG-tagged ERdj5 was modified with mPEG2000-mal in HEK293T cells treated with Tg or Tu for the indicated amount of time. Double or single asterisks denote modified ERdj5 bands. (B) Twenty-four hours after cotransfection of FLAG-tagged SERCA2b and Myc-tagged ERdj5/WT into HEK293 cells, the cells were treated with Tg or ionomycin for the indicated amount of time. The percentage binding of ERdj5 was normalized to that in untreated cells. (C) FLAG-SERCA2b was captured with FLAG-conjugated beads in lysates of cells transfected with FLAG-SERCA2b. Lysates of cells transfected with Myc-tagged ERdj5 were adjusted to contain the indicated concentration of CaCl2. The adjusted cell lysates were incubated with FLAG-SERCA2b-bound beads for the pull-down assay. ERdj5-Myc bound to SERCA2b was detected by immunoblotting with an anti-Myc antibody. Binding of ERdj5 with SERCA2b was quantified and shown below the panel.

ERdj5 regulates [Ca2+]ER by oligomer formation. (A) Relative scattering intensity derived from the monomeric component of ERdj5/SS or PDI at various calcium concentrations. (B) The Z-average, the mean diameter of ensemble particles in solution, of ERdj5/SS or PDI under various calcium concentrations. Values are the means ± SD of five independent experiments. (C) ERdj5-FLAG was captured with FLAG-conjugated beads in lysates of cells transfected with ERdj5-FLAG. Lysates of cells cotransfected with Myc-tagged ERdj5 and BiP/WT or the T37G mutant were adjusted to contain the indicated concentration of CaCl2. The adjusted cell lysates were incubated with ERdj5-FLAG-bound beads for the pull-down assay. After pull down with ERdj5-FLAG-bound beads, Myc-tagged ERdj5 bound to FLAG-tagged ERdj5 was detected by immunoblotting with an anti-Myc antibody. (D) Twenty-four hours after transfection of the indicated constructs into HEK293 cells, cells lysates containing 1 mM EGTA and titrated CaCl2 were prepared. Cell lysates were applied to a 10–40% sucrose density gradient and centrifuged. Each fraction was separated by SDS/PAGE for immunoblotting with the indicated antibodies.

BiP binding to ERdj5 prevents ERdj5 oligomer formation. (A) Twenty-four hours after cotransfection of FLAG-tagged SERCA2b, FLAG-tagged ERdj5/WT, and BiP into HEK293 cells, cell lysates were prepared for immunoprecipitation with an anti-FLAG antibody. Myc-tagged ERdj5 bound to FLAG-tagged SERCA2b was detected by immunoblotting with an anti-Myc antibody. (B) Twenty-four hours after cotransfection of BiP and ERdj5/WT or the H63A mutant into HEK293 cells, cell lysates containing 1 mM EGTA and 5 mM CaCl2 were prepared. Cell lysates were applied to a 10–40% sucrose density gradient and centrifuged. Each fraction was separated by SDS/PAGE for immunoblotting with an anti-Myc antibody. (C and D) SERCA2b activation mechanism via the reducing activity of ERdj5 depending on [Ca2+]ER. ERdj5 activates SERCA2b only at a lower [Ca2+]ER, whereas a higher [Ca2+]ER induces ERdj5 to form oligomers that are no longer able to interact with the pump. BiP exerts a regulatory role in the oligomerization of ERdj5 by binding to its J domain.

Homology models of SERCA2 provide a likely mechanism of the redox-dependent regulation of SERCA2b activity. (A) The Cys875–Cys887 pair and L5–L6 and L7–L8 loops in a SERCA2 homology model in the E1-2Ca state are highlighted. (B) Superposition of homology models of SERCA2 in the E2 (red), E1-2Ca (green), E1-2Ca-ATP (blue), and E2P (yellow) states. The homology model of SERCA2 was constructed for each state with SWISS-Model () using previously reported crystal structures of SERCA1a (Protein Data Bank ID: 3W5C for E2, 1SU4 for E1-2Ca, 3AR2 for E1-2Ca-ATP, and 3B9B for E2P) as templates. The transmembrane regions of these four SERCA2 models were superposed to minimize the root-mean-square deviation of their Cα atoms. Comparison of the models in different catalytic states suggests that the L7–L8 region of SERCA2 undergoes significant conformational changes during its catalytic cycle. (C) Conformational changes in the L7–L8 region upon reduction of the luminal Cys875–Cys887 disulfide bond. The homology models in the oxidized (cyan) and reduced (magenta) forms were constructed based on the crystal structures of both of the oxidized and the reduced forms of SERCA1a in the E1-2Ca-ATP state. The predicted position of the C-terminal tail of SERCA2b is shown in green dots. (D) Comparison of the relative position of the SERCA2 N domain in the E1-2Ca-ATP state. The homology models of the oxidized and reduced forms in the E1-2Ca-ATP state are shown in red and green, respectively. The model in the E1-2Ca form is shown in light brown.